Since work on this project begun in February 2017, we have generated a large panel of novel IFITM3 mutants to aid our study. Furthermore, we have developed systems using MLV-based vectors and adapted a pseudotyping procedure to study how various retroviral glycoproteins affect viral susceptibility to IFITM-mediated antiviral activities. Our data thus far suggest that IFITM3 exhibits great breadth with regard to its ability to inhibit retroviral particle infectivity. These findings suggest that these antiviral proteins may impose serious barriers to multiple mammalian retroviruses of medical importance in different species. Specifically, the presence of IFITM3 in virus-producing cells leads to decreased levels of envelope glycoprotein, which results in virus particles displaying very little glycoprotein needed to attach and perform fusion with cell targets. The first installment of this work is ready for submission and it demonstrates that IFITM3 can perform this activity when expressed ectopically or endogenously, as RNAi and CRISPR/Cas9-mediated gene knockout of IFITM3 in human cells and in murine cells result in longer half-lives of viral glycoproteins in cells. This indicates that IFITM3 negatively impacts the stability of viral glycoproteins via targeting to lysosomes. We also found that IFITM3 can target diverse viral glycoproteins for destruction in lysosomes, but that some are resistant, such as Ebolavirus glycoprotein. Furthermore, a number of cellular glycoproteins are unaffected by IFITM3. This finding reveals that IFITM3 utilizes a general mechanism to broadly inhibit the production of viral glycoproteins needed for virus fusion and spread. We have also identified a number of IFITM3 mutants that have lost this antiviral function. We are currently using structural homology to predict functional motifs to which these mutated sites belong, which will facilitate our understanding of how and why individual mutations disrupt activity. Together, these efforts will provide extensive insight into the function of IFITM proteins and will provide leverage for the development of new anti-HIV therapies.